CN105726888A - Application of Yiganle tablet to preparation of drugs for inhibiting mast cell leukemia cell CHMas proliferation - Google Patents

Application of Yiganle tablet to preparation of drugs for inhibiting mast cell leukemia cell CHMas proliferation Download PDF

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CN105726888A
CN105726888A CN201610187776.8A CN201610187776A CN105726888A CN 105726888 A CN105726888 A CN 105726888A CN 201610187776 A CN201610187776 A CN 201610187776A CN 105726888 A CN105726888 A CN 105726888A
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preparation
herba
chmas
diisopropylamine dichloroacetate
cell
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不公告发明人
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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Jinan Xinshidai Medicine Science and Technology Co Ltd
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    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
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    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying

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Abstract

The invention belongs to the technical field of traditional Chinese medicines and in particular relates to an application of a Yiganle tablet to preparation of drugs for inhibiting mast cell leukemia cell CHMas proliferation and a preparation method of the Yiganle tablet. The Yiganle tablet is prepared by using 200g of Chinese fevervine herb, 160g of giant knotweed rhizome, 200g of European verbena, 200g of snow of June herb, 200g of Chinese wedelia herb, 200g of rubus parvifolius, 170g of pink reineckea herb, 100g of Chinese mahonia stem and 70g of pilea notata as raw medicines and is prepared by adopting supercritical extraction, so that the content of emodin is greatly increased.

Description

The application in mast cell leukemia cell CHMas cell proliferation should be suppressed by diisopropylamine dichloroacetate sheet in preparation
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to a kind of suitable diisopropylamine dichloroacetate sheet and suppress the application in mast cell leukemia cell CHMas cell proliferation and the preparation method of suitable diisopropylamine dichloroacetate sheet in preparation.
Background technology
Suitable Ganle Granules standard No. WS-10148(ZD-0148)-2002, it is recorded in country's standard for traditional Chinese medicines compilation internal medicine liver and gall fascicle.It is made up as crude drug of fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, there is heat-clearing and toxic substances removing, effect of promoting the function of the gallbladder to alleviate jaundice.Acute or chronic hepatitis B caused by dampness-heat in the liver and gallbladder.
In prior art, not yet there is the suitable diisopropylamine dichloroacetate sheet report in the application suppressed in people's mast cell leukemia cell CHMas cell proliferation in preparation, also extract preparation aspect there are no suitable diisopropylamine dichloroacetate sheet and adopt the report of supercritical extraction, and traditional water decoction is boiled, the method for alcohol reflux, technique is coarse, backward, and impurity is many, causes that patient's consumption is excessive, be inconvenient to take, had a strong impact on this product and applied clinically.
Summary of the invention
Goal of the invention: it is an object of the invention to provide a kind of suitable diisopropylamine dichloroacetate sheet and suppress the application in mast cell leukemia cell CHMas cell proliferation in preparation.
Further object is that the preparation method that a kind of suitable diisopropylamine dichloroacetate sheet is provided.
It is an object of the invention to by following scheme realization:
The application in mast cell leukemia cell CHMas cell proliferation should be suppressed by diisopropylamine dichloroacetate sheet in preparation, described should be made up as crude drug of fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g by diisopropylamine dichloroacetate sheet, described the preparation method of diisopropylamine dichloroacetate sheet should comprise the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700-800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
Preferably, above-mentioned suitable diisopropylamine dichloroacetate sheet suppresses the application in mast cell leukemia cell CHMas cell proliferation in preparation, described the preparation method of diisopropylamine dichloroacetate sheet should comprise the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
In prior art, should be administered orally by Ganle Granules, a 10g, 3-4 time on the one.Suitable Ganle Granules dosage is big.The suitable every tablet weight 0.30g of diisopropylamine dichloroacetate sheet adopting the inventive method to prepare, only needs 2 every time, within 1st, takes 3-4 time.Dose is greatly reduced when having more active component.This conclusion can pass through following it have been experienced that.
The comparison of emodin content in suitable diisopropylamine dichloroacetate sheet prepared by test one, distinct methods
L, instrument and the reagent present invention should diisopropylamine dichloroacetate sheets: prepare by embodiment 1 method, use 1500g crude drug, extracted make 200, every tablet weight 0.30g.Former should Ganle Granules, according to WS-10148(ZD-0148) prepared by-2002 standard methods.Agilent1200 high performance liquid chromatograph；METTLERAE240 electronic analytical balance；Rheum emodin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute).
2, method
Measure according to high performance liquid chromatography (Chinese Pharmacopoeia the 4th note on the use 15 of version in 2015).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；Methanol-0.1% phosphoric acid solution (80: 20) is mobile phase；Detection wavelength is 437nm.Number of theoretical plate calculates by rheum emodin peak should be not less than 4000.
It is appropriate that the preparation precision of reference substance solution weighs rheum emodin reference substance, adds methanol and makes every 1ml solution containing 50 μ g, to obtain final product.
The preparation of the need testing solution of the suitable diisopropylamine dichloroacetate sheet of the present invention takes the suitable diisopropylamine dichloroacetate sheet 5 of the present invention, finely ground, takes 0.12g, accurately weighed, put in tool plug conical flask, add dehydrated alcohol 20ml, supersound process (power 250W, frequency 25kHZ) 1 hour, let cool, filter, medicinal residues absolute ethanol washing 4 times, each 10ml, merging filtrate and cleaning mixture, be evaporated, and residue adds dilute hydrochloric acid 10ml makes dissolving, add chloroform 20ml, it is heated to reflux 30 minutes, lets cool, move in separatory funnel, with a small amount of chloroform container, it is incorporated in separatory funnel；Dividing and take chloroform solution, acid solution adds chloroform 2 times, each 10ml, and combined chloroform liquid is evaporated, and residue precision adds methanol 10ml, weighed weight, and slight fever makes dissolving, lets cool, more weighed weight, adds methanol and supplies the weight of less loss, shakes up, and filters, takes subsequent filtrate, to obtain final product.
The preparation of reference product need testing solution takes the suitable Ganle Granules 20g of this product comparison, finely ground, takes 4g, accurately weighed, put in tool plug conical flask, add dehydrated alcohol 20ml, supersound process (power 250W, frequency 25kHZ) 1 hour, let cool, filter, medicinal residues absolute ethanol washing 4 times, each 10ml, merging filtrate and cleaning mixture, be evaporated, and residue adds dilute hydrochloric acid 10ml makes dissolving, add chloroform 20ml, it is heated to reflux 30 minutes, lets cool, move in separatory funnel, with a small amount of chloroform container, it is incorporated in separatory funnel；Dividing and take chloroform solution, acid solution adds chloroform 2 times, each 10ml, and combined chloroform liquid is evaporated, and residue precision adds methanol 10ml, weighed weight, and slight fever makes dissolving, lets cool, more weighed weight, adds methanol and supplies the weight of less loss, shakes up, and filters, takes subsequent filtrate, to obtain final product.
Algoscopy precision respectively draws reference substance solution and each 5 μ l of need testing solution, injects chromatograph of liquid, measures, to obtain final product.
3, result
It is shown that the present invention should the content of rheum emodin be 0.95-1.86mg/ sheet in diisopropylamine dichloroacetate sheet；And former should Ganle Granules be 0.91mg/ bag (every bag containing granule 10g) at the content of rheum emodin, each serving 2-4 times that emodin content is former granule 10g content of consumption 2, when dose reduces, emodin content improves a lot.
The studies above shows, adopts suitable diisopropylamine dichloroacetate sheet prepared by preparation method of the present invention, and active constituent content is significantly larger than WS-10148(ZD-0148) the suitable Ganle Granules prepared of method recorded of-2002 standards.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further described, so that those skilled in the art knows more about the present invention, but this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to Examples below, and all technology realized based on foregoing of the present invention belong to the scope of the present invention.
Embodiment 1
Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
After testing, in finished product, the content of rheum emodin is 1.85mg/ sheet.
Embodiment 2
Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 4% that entrainer accounts for the percent by volume of total extractant, extracting pressure 15MPa, temperature 30 DEG C, CO2Flow 1ml/g crude drug min, extraction time 900min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
After testing, in finished product, the content of rheum emodin is 1.21mg/ sheet.
Embodiment 3
Take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 6% that entrainer accounts for the percent by volume of total extractant, extracting pressure 30MPa, temperature 60 C, CO2Flow 3ml/g crude drug min, extraction time 800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
After testing, in finished product, the content of rheum emodin is 0.95mg/ sheet.
Embodiment 4: suitable diisopropylamine dichloroacetate sheet suppresses the experimentation data of people's mast cell leukemia cell CHMas cell proliferation
1. experiment material
1.1 experiment cell strains
People's mast cell leukemia cell CHMas cell, Shandong University's laboratory cell storehouse, DMEM+10%FBS cellar culture.
1.2 Experimental agents
Drugs: the present invention should diisopropylamine dichloroacetate sheet: prepare by embodiment 1 method.
Medicinal liquid liquid storage: weigh 100mg should diisopropylamine dichloroacetate sheet, be dissolved in 5ml dehydrated alcohol, 0.2 μm of frit, 500 μ ldoff pipe subpackages ,-20 DEG C of storages, simultaneously 0.2 μm of frit dehydrated alcohol prepares against the use of matched group.
1.3 experiment reagents
DMEM (GIBCO company Cat.No.12100-061Lot.No.758137)；Hyclone (Tian Hang bio tech ltd, Zhejiang Lot.No.100419)；NaHC03(Shanghai hundred million chemical reagent company limited Cat.No.11810-033Lot.No.1088387 of a specified duration)；Trypsin(AMRESCO company)；EDTA(AMRESCO company)；PenicillinGSodiumSalt(AMRESCO company 1)；StreptomycinSulfate(AMRESCO)；Dehydrated alcohol (Zibo Ya Dulan Trade Co., Ltd.)；MTT (Biosharp lot number: 0793): PBS(laboratory autogamy)；
1.4 experiment equipments
Lycra inverted microscope (Germany Leica model: DMIL)；Visible-ultraviolet light microwell plate detector (MD company of U.S. model: SPECTRAMAX190)；C02Incubator (FORMA model: 3111)；Super-clean bench (safe and sound company of Su Jing group manufactures model: SW-CJ-ZFD)；Pure water instrument (Sprlng company of U.S. model: S/N020579)；Accurate pipettor (Gilson Inc of France model: P2)；Electronic balance (Sai Duolisi company limited of Germany model: BT323S)；Full-automatic high-pressure autoclave (SANYO company of Japan model: MLS-3020)；Table electrothermal air dry oven (Shanghai precision experimental facilities company model: DHG9123A)；Refrigerator (Siemens Company's model: KG18V21TI)；Liquid nitrogen container (CBS model: 2001)；Low speed centrifuge (Anting Scientific Instrument Factory, Shanghai's model: KA-1000)；0.2 μm of filter (MILLIPORE model: SLGP033RB)；1cm culture dish (NEST company), 96 well culture plates (NEST company)；Cell counting count board；Centrifuge tube, pipet, Tips are some.
2. experimental technique
1) CHMas cell DMEM+10%FBS is in 37 DEG C, 5%C02Carry out cellar culture (10cm culture dish), when Growth of Cells to logarithmic (log) phase, collect cell, discard culture fluid, PBS fine laundering 3 times, add 3ml0.25% trypsin-0.04%EDTA, after 37 DEG C of digestion 2min, it is added thereto in 5ml complete medium and reaction, is proceeded in centrifuge tube after piping and druming cell, 1000rpm is centrifuged 5min, adjusts concentration of cell suspension 3 × 104Individual/ml.
2) entering in 96 well culture plates by cell kind, every hole adds cell suspension 180 μ l, culture plate put in cell culture incubator (37 DEG C, 5%C02) cellar culture.
3) according to cell growth status, general long to 50%-70%, add suitable diisopropylamine dichloroacetate sheet solution, continue to cultivate 24h.
4) add 20 μ lMTT solution (5mg/ml, i.e. 0.5%MTT) after 24h, continue to cultivate 4h.
5) after 4h, buckle method removes supernatant, pats dry gently with absorbent paper, and every hole adds 200 μ l dimethyl sulfoxide, puts low-speed oscillation 10min on shaking table, makes crystal fully dissolve.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm place.
6) arranging background (being not added with cell, only add culture fluid), control wells (cell, the medicine dissolution medium of same concentrations, culture fluid, MTT, dimethyl sulfoxide), often group sets 6 multiple holes simultaneously.
7) suppression ratio of cell is represented by result with medicine: cell proliferation suppression ratio (%)=(control wells OD value-dosing holes OD value), control wells OD value × 100%.Experiment repeats 3 times.
3. statistical disposition
Adopting the correlation analysis in MicrosoftExcel2007 software and Studentt inspection, data represent with mean ± S.D..
4. experimental result
Statistical result showed after mtt assay experiment, compare with matched group, when dosage reaches 5mg/ml, to CHMas cell inhibitory effect variant (P < 0.05), dosage this difference when 10mg/ml has significance (P < 0.01), has pole significant difference (P < 0.001) when dosage reaches 15-20mg/ml.
The suitable diisopropylamine dichloroacetate sheet of table 1 is to CHMas cell inhibitory effect influence research (X ± SD)
Group Drug level (mg/ml) Suppression ratio (%)
Matched group 0 0
1 5 12.56±4.56
2 10 23.05±6.03*
3 15 32.65±7.98**
4 20 40.46±9.46**
Note: compare with matched group, * P < 0.01；**P<0.001.
5. experiment conclusion
The suitable diisopropylamine dichloroacetate sheet of the present invention can suppress CHMas cell proliferation, reduces the Growth of Cells number of CHMas cell, and this effect is dose dependent.

Claims (2)

1. should suppress the application in mast cell leukemia cell CHMas cell proliferation in preparation by diisopropylamine dichloroacetate sheet, described suitable diisopropylamine dichloroacetate sheet is by fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g makes as crude drug, it is characterized in that, described should the preparation method of diisopropylamine dichloroacetate sheet comprise the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 4-6%, extracting pressure 15-30MPa that entrainer accounts for the percent by volume of total extractant, temperature 30-50 DEG C, CO2Flow l-3ml/g crude drug min, extraction time 700-800min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
2. suitable diisopropylamine dichloroacetate sheet according to claim 1 suppresses the application in mast cell leukemia cell CHMas cell proliferation in preparation, it is characterized in that, described the preparation method of diisopropylamine dichloroacetate sheet should comprise the steps: to take fevervine 200g, Rhizoma Polygoni Cuspidati 160g, Herba Verbenae 200g, Serissa foetida 200g, Herba Kalimeridis grass 200g, Rubus Parvifolius L. 200g, Herba Reineckeae Carneae 170g, Caulis Mahoniae 100g, Rhizoma Belamcandae 70g, join CO2In supercritical extraction device, ethanol is as entrainer, and it is 5% that entrainer accounts for the percent by volume of total extractant, extracting pressure 25MPa, temperature 40 DEG C, CO2Flow 2ml/g crude drug min, extraction time 750min, obtain supercritical extract, supercritical extract added starch, 70% ethanol granule, dry, and tabletting makes 200, every tablet weight 0.30g.
CN201610187776.8A 2016-03-29 2016-03-29 Application of Yiganle tablet to preparation of drugs for inhibiting mast cell leukemia cell CHMas proliferation Withdrawn CN105726888A (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN1785351A (en) * 2005-11-03 2006-06-14 贵州天赐医药生物有限公司 Soft capsule for trenting acute, chronic hepatitis B and its preparation method

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CN1785351A (en) * 2005-11-03 2006-06-14 贵州天赐医药生物有限公司 Soft capsule for trenting acute, chronic hepatitis B and its preparation method

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Application publication date: 20160706